Nano-scale hydroxyapatite (HAp) was prepared by combining co-precipitation with microemulsion method, which exhibited strong adsorption for DNA due to its excellent biocompatibility and particular adsorbability. DNA and HAp could be modified onto glassy carbon electrode (GCE) by the simple and convenient “tip-coating” method. Cyclic voltammetry was used to investigate the interaction of DNA immobilized on the HAp film with vitamin B12 (VB12). The existence of DNA led to the decrease of reduction current of VB12. Both the electron transfer coefficient (α) and the standard rate constant (ks) were different obtained on GCE and dsDNA/HAp/GCE, which indicated the formation of an electrochemical inactive super molecular complex DNA-nVB 12 . The equilibrium constant of this complex was calculated to be 5.35 × 10 5 mol·L -1 and the binding number between DNA and VB12 of the complex were determined to be one.